ELISA (Enzyme Linked Immuno Sorbent Assay) test procedures have been developed for the detection of organic substances in blood and other bodily and biological fluids.
The ELISA technique is based upon three primary principles; first, the ability of proteins to bind to plastic; the high affinity and specificity of antigen/antibody reactions and the ability of enzymes to modify a substrate. ELISA assay procedures are well known, and will not therefore be described in great detail herein. Briefly however, a plastic plate having typically 96 individual plastic wells is the receptacle for the fluid sample. Each of the 96 wells is coated with an antibody and an antigen specific to the antibody is bound thereto to hold the antigen in place. A second antibody specific to the same antigen is added and bonded thereto. The second antibody has been covalently conjugated prior to its introduction with an enzyme which will react with a substrate to cause a colour change therein given the right environmental conditions. The degree of colour change in the substrate is proportional to the amount of the biological substance such as an antigen sought to be detected. By passing filtered light through the substrate and measuring its degree of absorption by the substrate using an ELISA reader, one can calculate the concentration of the biological substance therein as a function of the amount of absorbed light, which is assigned a numerical value or index. The greater the colour intensity of the substrate, the greater the absorption of light.
The use of ELISA techniques has increased greatly having regard to the procedure's relative simplicity, speed, reliability, sensitivity and as a means of avoiding the use of radioactive assays.
The disadvantages of ELISA readers that measure light absorption are several. The readers themselves are delicate and sensitive and require constant adjustment to maintain optimal sensitivity. The linear response of the readers falls within a very narrow range so that each test requires large numbers of dilutions so that at least one or two of such dilutions falls within the linear range of the instrument. A relatively intense light source is required making it more difficult to obtain a portable unit having regard to the inherent power requirements of the system, and of course the plastic lens at the bottom of each well must pass the light without distortion, necessitating the use of relatively expensive optical grades of plastic which further adds to the costs. The readers themselves are relatively expensive (prices range from $6000. for relatively simple models, up to $28,000. for units with computerized data management and analytical capabilities) and some existing readers perform only one light measurement at a time, making the reading of a 96 well plate a relatively time-consuming process.